Increasing hen eggshell thickness with 25-hydroxycholecalciferol compounds

ABSTRACT

THE ADMISISTRATION OF 25-HYDROXYCHOLECALCIFEROL, 21, 25DIHYDROXYCHLOECALCIFEROL, 25,26-DIHYDROXYCHLECALCIFEROL, 1,25-DIHYDROXYCHOLECALCIFEROL, 25-DHYDRXYDIHYDROTACHYSTEROL3, 25-HYDROXYERGOCALCIFEROL AND THEIR ACYLATES TO HENS INCRESES THE THICKNESS OF THE EGGSHELLS. COMPOSITIONS AND METHODS ARE PROVIDED.

UfiitedStates' Patent Oflice 3,823,237 Patented July 9, 1974 3,823,237 INCREASING HEN EGGSHELL THICKNESS WITH 25 HYDROXYCHOLECALCIFEROL COMPOUNDS Fred Richard Frank, Portage, Mich., assignor to The Upjohn Company, Kalamazoo, Mich.

No Drawing. Continuation of abandoned application Ser. No. 210,232, Dec. 20, 1971. This application Jan. 17, 1972, Ser. No. 218,480

Int. Cl. A61]; 27/00 US. Cl. 424-236 4 Claims ABSTRACT OF THE DISCLOSURE The administration of 25-hydroxycholecalcifero1, 21,25- dihydroxycholecalciferol, 25,26-dihydroxycholecalciferol, 1,2S-dihydroxycholecalciferol, 25-hydroxydihydrotachysterol ZS-hydroxyergocalciferol and their acylates to hens increases the thickness of the eggshells. Compositions and methods are provided.

CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part application of my earlier filed copending application, Ser. No. 210,232 filed on Dec. 20, 1971, now abandoned.

THE BACKGROUND The poultry industry has recently made significant advances in dealing with many of its problems. However, one problem area which has met with little success is the effort to decrease the incidence of broken hens eggs. Approximately 58% of eggs laid by hens are prematurely broken with a loss to egg producers of up to 100 million dollars or more per year. Generally, the reason for the premature breakage appears to be insuflicient calcification of the egg shell, a condition which produces an egg shell which is too thin to withstand forces exerted upon it. It is well known that Vitamin D affects the shell thickness of hens eggs. However, Vitamin D has little or no effect on the thickness of egg shells from some hens. Furthermore, the activity of Vitamin D for increasing shell thickness reaches a plateau which does not show an increase even at extremely high dosages of Vitamin D Recently an active metabolite of Vitamin D has been isolated. This metabolite, the 25-hydroxy derivative of Vitamin D generically known as 25-hydroxycholecalciferol, and referred to hereafter as 25-HCC, has been shown to be more active than Vitamin D in the rat line rickets cure assay and in the rickets prevention assay in young chicks. These tests were made on apparently normal rats and chicks who were fed abnormal diets prior to the experiment so as to induce a type of disease condition in order to demonstrate that ZS-HCC has Vitamin D -like activity. Since other suspected Vitamin D metabolites have only been active in one of these species, this was strong evidence that a true active metabolite of Vitamin D was isolated. Additionally, a more rapid onset of intestinal transport of calcium from the mucosal to serosal side and of bone mobilization in rats is shown with administration of 25-HCC.

However, no particular tests using 25-HCC in mature egg-laying chickens or other species are disclosed in the prior art. Perhaps of even greater significance, only problems dealing with bone calcification such as rickets and osteomalacia, or general membrane calcium transport, such as cattle milk fever and hypoparathyroidism in humans have been dealt with by the art.

The increasing of hen egg shell thickness and the curing or preventing of bone conditions such as rickets and osteomalacia proceed by different physiological mechanisms of action. The substance which is responsible for increasing shell thickness is calcium carbonate. The substance deposited which aids in treating the various bone diseases is calcium hydroxyapatite, a complex mineral-like salt of phosphorus, oxygen, and calcium which is chemically unrelated to carbonate. Furthermore, the organic matrices in which each of these substances is deposited differ radically from each other. Egg shell organic matrix is comprised of noncellular amorphous sheets of protein. The bone organic matrix is formed, living tissue capable of reproducing itself.

BRIEF SUMMARY OF THE INVENTION It has been found that 25-HCC and related compounds to be described hereinafter have the capacity to increase the thickness of hens egg shells. When administered to hens, these compounds increase the thickness of shells from a substantial number of hens whose egg shells exhibit a thickness of less than or equal to 0.013 inch, a thickness which is considered to be critical since most breakage occurs in egg shells with a thickness less than 0.013 inch. Surprisingly, this egg shell thickening can be observed in approximately double the number of hens whose egg shells are thickened by administering the same quantities of Vitamin D without an apparent decrease in egg production. The fact that these outstanding results are obtained in the plateau region of Vitamin D dosage is even more surprising, since the effect is not solely due to the difference in activity of Vitamin D and 25-HCC.

Furthermore, even where egg shell thicknesses are above 0.013 inch and it may be assumed that all the hens metabolic processes relating to egg calcification are functioning properly, the administration of ZS-HCC and related compounds at the Vitamin D dosage plateau level induced a thickening of egg shells from a substantially greater number of chickens than when employing the same quantities of Vitamin D It is also of significance that the increase in shell thickness in both of the situations above, after the administration of 25-HCC, was at least equal to the increase in shell thickness observed after administration of Vitamin D Therefore, in accordance with this invention, a feed formulation for increasing the thickness of hens egg shells is disclosed comprising a chicken feed in combination with an egg shell thickening effective but non-toxic quantity of a compound selected from the group consisting of where R=H, or acyl where acyl is an acyl radical of normal or isomerized alkane carboxylic acid of from 2 to about 8 carbon atoms and R, R, and R can be H, OH, or O acyl wherein acyl is defined as above, but that at least two of R, R", and R must be hydrogen and that if R, R, or R is OH then R must be H, and if R, R, or R is O acyl, acyl defined as above, then R must be the same acyl.

A further aspect of this invention is the process for increasing the shell thickness of eggs from hens comprising feeding the hens a chicken feed comprising the chicken feed in combination with an egg shell thickening effective but non-toxic quantity of a compound selected from the group of compounds disclosed above with reference to composition components.

DETAILED DESCRIPTION OF THE INVENTION The compounds employed in the compositions and uses of those compositions are prepared by methods known to the art. The unacylated compounds of the invention have been prepared in the following references:

Formula A:

25-HCC R=R=R"=R=H U.S. Patent 3,565,924

21, 25 Dihydroxyeholecalcilerol-R=R=R'=H, R"=OH Biochemistry 9;14=2,917-2,922, 1970 25, 26-..- Dihydroxyeholeealciferol-R=R=R=H, R=OH Biochemistry 9:24:4,776-4,780, 1970 1, 25 Dihydroxychplecaleiferol-R=R"=R'=H, R=OH Bioehenustry 10: 14:2,799-2,804, 1971 Formula B: 25.- Hydroxydlhydrotaehysteroh-R=H Federation Proceedings 29:2:687, 1970 Formula C: 25.. HydroxyergocalciferolR=H Biochemistry 8 9 3,5153,5l9,1969

The acylated compounds of this invention can be prepared by taking any of the above unacylated compounds and reacting it with the acid anhydride or chloride of the two to eight carbon atom hydrocarbon carboxylic acid desired according to the method disclosed in Example 16 of Babcock and Campbell, Belgian Pat. No. 747,492. Il-

lustrated reactants and respective products are the following:

2S-hydroxycholecalciferol treated with acetic anhydride gives 2S-hydroxycholecalciferol 3-acetate 25-hydroxycholecalciferol treated with butyryl chloride gives 2S-hydroxycholecalciferol 3-butyrate 25-hydroxycholecalciferol treated with octanoyl chloride gives 2S-hydroxycholecalciferol 3-octanoate 2l,25-dihydroxycholecalciferol treated with acetic anhydride gives 21,25-dihydroxycholecalciferol 3,21-diacetate 21,2S-dihydroxycholecalciferol treated with isobutyryl chloride gives 21,2S-dihydroxycholecalciferol 3,21diisobutyrate 25,26-dihydroxycholecalciferol treated with propionic anhydride gives 25,26-dihydroxycholecalciferol 3,26-dipropionate 1,25-dihydroxycholecalciferol treated with pentanoic anhydride gives 1,25-dihydroxy cholecalciferol 1,3-dipentanoate 25-hydroxydihydrotachysterol, treated with acetic anhydride gives 2S-hydroxydihydrotachysterol 3-acetate 25 hydroxyergocalciferol treated with acetic anhydride gives ZS-hydroxyergocalciferol 3-acetate It is preferred to use the hydrated forms of metabolites whenever possible. For example, the hydrate of 25-HCC is preferred over the non-hydrate of 25-HCC. The method of preparation is disclosed in Belgian Pat. No. 747,492, Example 8.

The chicken feed into which the compounds are combined is any standard diet fed an egg laying hen. An example of such a diet is given below:

Layer formulation (fed after 20 weeks of Ingredients: age) (pounds) Corn meal, No. 2 yellow, ground 667.5 Soybean meal, 50% protein 100 Fish meal, 65% protein 15 Meat and bone scraps, 50% protein 75 Corn distillers dried solubles 50 Alfalfa meal, 17% protein l5 Limestone, ground 70 Salt, iodized 2.5 DL-Methionine 0.5 Vitamin premix 5 1 Calculated composition:

Protein, percent 17.2 Calcium, percent 3.4

The calcium level for egg laying hens is well known and important for the calcification of egg shells. Generally from about 2.5 to about 5.5% calcium, calculated as weight of calcium per weight of feed formulation is required, preferably from about 3.0 to about 4.0%. When levels of calcium at the upper end of the range are employed, a diet with increased energy and protein concentration can be used.

Prior to combination of the active compound with the feed, it is preferable to coat the compound to prevent degradation. This is particularly desirable when employing the non-hydrated form of the compound. Any of the standard vitamin coatings can be employed. For example, the crystalline compound can be coated with a solid antioxidant in an amount suflicient only to coat the surface of the crystals with the substance and then drying the crystals as in US. Pat. No. 3,110,783, or a solid or liquid composition containing the compounds can be stabilized by addition of standard antioxidants such as a tocopherol, propyl gallate, butylated hydroxy anisole, and butylated hydroxy toluene.

The active compound can be combined with the chicken feed formulation as the pure compound or in combination with other feed components, in its crystalline form, in

solution, or in suspension, as long as the compound is uniformly present throughout the feed compositions.

The compound should be present in the feed formulation in egg shell thickening but non-toxic quantities. As much compound can be used as desired as long as toxic effects are not observed. The preferred quantity of compound is that minimum amount which induces the desired egg shell thickening results. Quantities of 25-HCC of from about 5 g. to about 20 g./lb. of feed formulation have induced egg shell thickening results as shown in Example 1.

The feed formulation of this invention is fed to hens on a normal dietary intake and desired egg shell thickening is observed.

The following examples are illustrative of the invention and are not to be construed as limiting the invention.

Example 1 150 Hyline strain SCWL hens fed with a diet including a commercially available form of stabilized Vitamin D and producing eggs with shells varying in thickness from less than 0.011 to greater than 0.015 inch, are evenly distributed among six treatment groups so that each treatment group contained all representative values of shell thickness. To a basal non-Vitamin-D supplemented feed formulation is added daily either 25-HCC or Vitamin D This diet is then fed to hens. The daily formulation was done to avoid vitamin degradation. Shell changes and rate of production are determined weekly. The chickens are on this schedule for ten weeks.

The results from this experiment show that shell thicknesses increased after administration of 25-HCC or Vitamin D from 0.001 to 0.003 inch. The increase in shell thickness of eggs from hens treated with 25-HCC was at least as good as from hens treated with Vitamin D Egg production appears to be unchanged after treatment with ZS-HCC.

Tables I and II present data with regard to improving shell thickness in eggs from a given number of hens after administration of 25-HCC or Vitamin D Where the egg shells are initially greater than 0.013 inch, the improvement of shell thickness when using 25-HCC is even more pronounced. The number of hens whose shell thickness increased after administration of ZS-HCC was approximately two and a half that of the hens treated with Vitamin D Since these were seemingly normal hens, the results cannot be explained by any theory now known.

Although 5 pg. of ZS-HCC was the smallest quantity employed in this experiment, this does not imply that desired egg shell thickening results cannot be achieved with smaller quantities. Likewise quantities above 20 g. could be equally or perhaps even more effective without showing observable toxic efiects.

Example 2 A calcium source such as oyster shells, physically separated from the chicken feed formulation, and impregnated with sufiicient 25-HCC to provide 3 g. of ZS-HCC per chicken per day is fed to chickens. The chickens ingest the oyster shell and absorb the 25-HCC from the digestive tract. Desired egg shell thickening is observed in subsequently laid eggs.

Examples 3 and 4 illustrate additional methods of administering the active compound to egg laying hens.

Example 3 A quantity of 25-HCC sufiicient to administer to a chicken about 3 g. of 25-HCC per day, assuming average water intake per day of the chicken, is metered into the chickens water supply. To facilitate proper dispersion, the 25-HCC can be micronized if desired and a surfactant such as polysorbate 80, see Merck Index, can be metered into the water supply. The chickens ingest their normal quantity of water including the 25-HCC Desired egg shell thickening is observed in subsequently laid eggs.

Example 4 A quantity of ZS-HCC suificient to provide a chicken with days supply of 25-HCC is combined with an inert carrier such as a mineral oil and injected subcuta- TABLE I Comparison of vitamin D and 254106 efiects on hens laying eggs 0.013 inch thick Number hens Percent Producing With oi hens shells improved with shell Chicken g./lb. 0. 013 c improvegroup Compound 01 teed Formulation inch thick fication ment Vitamin D3. 10 Crystalline 16 6/16 37. 5 10 Propylene glycol- 14 6/14 42. 8 5 d0 12 9/12 75.0 10 do 1 8/11 72.7 10 Crystalline 15 13/15 86. 6

20 Propylene glycol. 11 9/11 81. 8

TABLE II Comparison of vitamin D and 25-HCC effects on hens laying eggs 0.013 inch thick Number hens Percent Producing With at hens shells improved with shell Chicken pg.llb. 0. 013 calciimprovegroup Compound 0t feed Formulation inch thick fication ment 1 Vitamin D 10 Crystalline 6 1/6 17. 0 do 10 Propylene glycol. 9 2/9 22. 0 5 do 7 4/7 56. 5 10 Crystalline 7 4/7 56. 5 10 Propylene glyc 6 3/6 50. 0 20 do 8 5/8 62. 5

neously into the web of the wing. The ZS-HCC is slowly released and absorbed. Desired egg shell thickening is observed in subsequently laid eggs.

Example 5 In a similar manner as exemplified in Examples 1, 2, 3,

and 4, 21,25-dihydroxycholecalciferol, 25,26-dihydroxycholecalciferol, 25-hydroxyergocaliferol, ZS-hydroxydihydrotachysterol and 1,25-dihydroxycholecaliferol are administered to egg laying hens and results similar to thos of Examples 1, 2, 3, and 4 are obtained.

Example 6 In a similar manner as exemplified in Examples 1, 2, 3 and 4, 25-hydroxycholecaliferol 3-acetate, 25-hydroxycholecaliferol 3-butyrate, 2S-hydroxycholecalciferol 3- octanoate, 2l,25-dihydroxycholecalciferol 3,21-diacetate, 2l,25-dihydroxycholecalciferol 3,21-diisobutyrate, 25,26 dihydroxycholecalciferol 3,26 dipropionate, 1,25 dihydroxycholecalciferol 1,3-dipentanoate, 25-hydroxydihydrotachysterol 3-acetate, 25-hydroxyergocalciferol 3 acetate are administered to egg laying hens and results similar to those of Examples 1, 2, 3, and 4 are obtained.

When employing an acylate the dosage should be related to its unacylated parent molecule on a molar basis. -For example, approximately 1.6 g. of 21,25-dihydroxycholecalciferol 3,2l-dioctanoate is equivalent to 1.0 ,ug. 2l,25-dihydroxycholecalciferol.

I claim:

1. The method for increasing the thickness of egg shells from egg laying hens comprising administering to said egg laying hens an effective non-toxic egg shell thickening amount of -hydroxycholecalciferol and acylates-thereof where the acyl is an acyl radical of normal or isomerized alkane carboxylic acid of from two to eight carbon atoms, inclusive, in combination with a chicken feedformulation having from about 2.5 to about 5.5% calcium per pound of chicken feed.

2; The method of Claim 1 wherein the compound is 25- hyd'roxycholecalciferol.

3. The method of Claim 2- wherein there is from about 5 ,ug. to about 20 ',ug.- of compound per pound of feed.

4. The method of Claim 3 wherein the 25-hydroxycholecalciferol is in its hydrated form. p

References Cited UNITED STATES PATENTS 3,565,924 2/1971 De Luca et al. 260397.2 3,445,572 5/ 1969 -Machlin 424-264 3,639,596 2/1972 De Luca et al. 424236 3,646,203 2/ 1972 De Luca et al. 424-236 ALBERT T. MEYERS, Primary Examiner F. E. WADDELL, Assistant Examiner 

